This proposal concerns mammalian cell genetics and the interaction of tumor virus DNA with that of the host cell. The long-range goal of the research is the development of an efficient, reproducible means of introducing into mammalian cells prokaryotic genes such that they can be stably incorporated and expressed. The steps required in the development of such a system are identification of a suitable gene and cell system; isolation of a large amount of a DNA fragment containing the gene of interest; linkage of this DNA in vitro to a molecular vehicle, such as SV40 DNA; infection of mammalian cells with the DNA; and detection of the expression of the prokaryotic gene. The specific objectives of the proposed research are directed toward two aspects of the overall problem, namely the uptake and expression of DNA by mammalian cells and the isolation of a suitable prokaryotic gene: (1) BSC-1 cells (permissive for simian virus 40) and 3T3 cells (nonpermissive) will be used to study the uptake and expression of SV40 DNA in an attempt to improve the rather inefficient techniques presently available. Uptake will be measured by the use of radiolabeled DNA and expression by assays for infectivity in BSC-1 cells and for transforming activity on 3T3 cells. The experiments will investigate physical and chemical parameters optimal for uptake; effects of drugs on uptake; use of cellular histones bound to SV40 DNA in vitro to protect against nuclease degradation in the cell; and infection of viable nuclei (isolated with cytochalasin B) followed by cell reconstruction. 2) Several bacterial enzymes will be screened to identify those which are not inhibited in vitro by mammalian cell extracts. Isolation of DNA containing the gene(s) of interest will be carried out by cleavage of DNA with restriction endonucleases followed by cloning and amplification of the specific fragments in E. coli by means of bacterial plasmids. The DNA will be purified and characterized by sedimentation and electrophoresis.